Influence of host phylogeographic patterns and incomplete lineage sorting on within-species genetic variability in Wigglesworthia species, obligate symbionts of tsetse flies

Vertical transmission of obligate symbionts generates a predictable evolutionary history of symbionts that reflects that of their hosts. In insects, evolutionary associations between symbionts and their hosts have been investigated primarily among species, leaving population-level processes largely unknown. In this study, we investigated the tsetse (Diptera: Glossinidae) bacterial symbiont, Wigglesworthia glossinidia, to determine whether observed codiversification of symbiont and tsetse host species extends to a single host species (Glossina fuscipes fuscipes) in Uganda. To explore symbiont genetic variation in G. f. fuscipes populations, we screened two variable loci (lon and lepA) from the Wigglesworthia glossinidia bacterium in the host species Glossina fuscipes fuscipes (W. g. fuscipes) and examined phylogeographic and demographic characteristics in multiple host populations. Symbiont genetic variation was apparent within and among populations. We identified two distinct symbiont lineages, in northern and southern Uganda. Incongruence length difference (ILD) tests indicated that the two lineages corresponded exactly to northern and southern G. f. fuscipes mitochondrial DNA (mtDNA) haplogroups (P = 1.0). Analysis of molecular variance (AMOVA) confirmed that most variation was partitioned between the northern and southern lineages defined by host mtDNA (85.44%). However, ILD tests rejected finer-scale congruence within the northern and southern populations (P = 0.009). This incongruence was potentially due to incomplete lineage sorting that resulted in novel combinations of symbiont genetic variants and host background. Identifying these novel combinations may have public health significance, since tsetse is the sole vector of sleeping sickness and Wigglesworthia is known to influence host vector competence. Thus, understanding the adaptive value of these host-symbiont combinations may afford opportunities to develop vector control methods.     

Dynamics of multiple symbiont density regulation during host development: tsetse fly and its microbial flora

Symbiotic associations often enhance hosts' physiological capabilities, allowing them to expand into restricted terrains, thus leading to biological diversification. Stable maintenance of partners is essential for the overall biological system to succeed. The viviparous tsetse fly (Diptera: Glossinidae) offers an exceptional system to examine factors that influence the maintenance of multiple symbiotic organisms within a single eukaryotic host. This insect harbours three different symbionts representing diverse associations, coevolutionary histories and transmission modes. The enterics, obligate mutualist Wigglesworthia and beneficial Sodalis, are maternally transmitted to the intrauterine larvae, while parasitic Wolbachia infects the developing oocyte. In this study, the population dynamics of these three symbionts were examined through host development and during potentially disruptive events, including host immune challenge, the presence of third parties (such as African trypanosomes) and environmental perturbations (such as fluctuating humidity levels). While mutualistic partners exhibited well-regulated density profiles over different host developmental stages, parasitic Wolbachia infections varied in individual hosts. Host immune status and the presence of trypanosome infections did not impact the steady-state density levels observed for mutualistic microbes in either sex, while these factors resulted in an increase in Wolbachia density in males. Interestingly, perturbation of the maternal environment resulted in the deposition of progeny harbouring greater overall symbiont loads. The regulation of symbiont density, arising from coadaptive processes, may be an important mechanism driving inter-specific relations to ensure their competitive survival and to promote specialization of beneficial associations.     

The nutritional importance of dietary blood components for reproduction in the tsetse fly, Glossina morsitans

Females of Glossina morsitans morsitans were fed diets of different composition and their performance in terms of survival, fecundity and offspring size, used as a basis for assessing the nutritional importance of the dietary constituents for reproduction. Diets comprised defibrinated bovine blood from which individual components had been removed, or various fractions of bovine blood mixed in different ratios and combinations.Flies fed serum-free diets (comprising saline-washed bovine erythrocytes) failed to reproduce and their ovaries were atrophied. Attempts were made to correct these symptoms by adding serum components to washed erythrocytes. Results showed that serum albumin and lipoproteins were vital for ovarian growth. The erythrocytes in defibrinated bovine blood could be replaced by preparations of haemoglobin. Dialysis against isotonic sodium chloride, removal of gamma globulins, removal of lymphocytes or heating to 55°C for 2 h did not alter the nutritional quality of defibrinated bovine blood. The qualitative importance of dietary albumin associated lipids and lipoproteins for oöcyte growth suggests a specialisation which may be peculiar to viviparous Diptera. Results are discussed in terms of the development of synthetic diets for tsetse.     

The control of receptivity and ovulation in the tsetse fly, Glossina morsitans

ABSTRACT. A rapid decline in receptivity of mated female Glossina morsitans morsitans Westwood is shown to depend on both physical and chemical stimuli associated with copulation. Radiolabelling revealed the transfer of substances from the male to the haemolymph of the female during copulation. Implantation of male tissues or their injection as homogenates into virgin females showed the chemical stimulus to come from the male accessory glands. Receptivity decreased in females mated to males with ejaculatory ducts severed or testes removed and also in females which had a glass bead inserted into their uterus and/or the tip of their abdomen covered with wax, suggesting that a physical stimulus inducing refractoriness is provided by distension of the uterus and/or stimulation of their terminalial setae. Exposing virgin females to daily short matings in which no male materials were transferred, confirmed this. Receptivity also declined slowly with age in unexposed virgin females. Transfusion of haemolymph from mated females (up to 11 days old) into virgins did not indicate the existence of a haemolymph-borne ovulation-inducing factor; apparently only physical stimuli from mating are involved in the induction of ovulation, and somehow prime the ovarian tissue so that it responds appropriately later when the egg has matured. Whether the stimulus is transmitted to the ovary neurally or hormonally is unknown.     

The major protein in the midgut of teneral Glossina morsitans morsitans is a molecular chaperone from the endosymbiotic bacterium Wigglesworthia glossinidia

Molecules in the midgut of the tsetse fly (Diptera: Glossinidiae) are thought to play an important role in the life cycle of African trypanosomes by influencing their initial establishment in the midgut and subsequent differentiation events that ultimately affect parasite transmission. It is thus important to determine the molecular composition of the tsetse midgut to aid in understanding disease transmission by these medically important insect vectors. Here, we report that the most abundant protein in the midguts of teneral (unfed) Glossina morsitans morsitans is a 60 kDa molecular chaperone of bacterial origin. Two species of symbiotic bacteria reside in the tsetse midgut, Sodalis glossinidius and Wigglesworthia glossinidia. To determine the exact origin of the 60 kDa molecule, a protein microchemical approach involving two-dimensional (2-D) gel electrophoresis and mass spectrometry was used. Peptide mass maps were compared to virtual peptide maps predicted for S. glossinidius and W. glossinidia 60 kDa chaperone sequences. Four signature peptides were identified, revealing that the source of the chaperone was W. glossinidia. Comparative 2-D gel electrophoresis and immunoblotting further revealed that this protein was localized to the bacteriome and not the distal portion of the tsetse midgut. The possible function of this highly abundant endosymbiont chaperone in the tsetse midgut is discussed.     

Ovulation, insemination and mating in the tsetse fly Glossina pallidipes

ABSTRACT. Analyses of the ovulation, insemination and mating of female Glossina pallidipes Austen, were carried out in the field and laboratory. In unmated females, ovulation was delayed until at least the fifteenth day of adult life. The predictable pattern of growth and development of eggs was used to age nulliparous females to the nearest day in the field. Inseminated females first appeared at 5–6 days old and most females became inseminated at 8–10 days of age. There were no significant differences in the age at insemination in Zimbabwe or Kenya. Ovulation had occurred in the field by 10–11 days old. Mating therefore occurs at or just before ovulation. Females were able to mate at up to 12 days old and still produce a viable larva from their first egg. In laboratory flies originating from Uganda, factors which reduced and minimized the effects of disturbance, maximized insemination rate, so that under the best conditions the age at mating was identical to that found in the field. Nevertheless, the insemination rate of Fl generation Zimbabwe flies reared from wild-caught females was negligible. The differences in the laboratory mating behaviour of the Zimbabwe flies and the flies from the long-established colony of Uganda origin, are considered to be due to wild G. pallidipes being easily disturbed under laboratory conditions, whereas colony breeding rapidly selects for passivity. The concept of gross differences in G. pallidipes mating behaviour between geographic areas in thus rejected.     

Lipophorin from the tsetse fly, Glossina morsitans morsitans.

1. Lipophorin was isolated from the haemolymph of adult tsetse fly, Glossina morsitans morsitans, by ultracentrifugation in a potassium bromide density gradient. 2. The tsetse fly lipophorin (Mr congruent to 600,000) has a density of congruent to 1.11 g/ml and consists of two apoproteins, apolipophorin-I (apoLp-I, Mr congruent to 250,000) and apolipophorin-II (apoLp-II, Mr congruent to 80,000), both of which are glycosylated as shown by staining with periodate-Schiff reagent. The protein complex is composed of 49% protein and 51% lipids. 3. The finding of lipophorin in tsetse fly haemolymph suggests that, although these flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements.     

Influence of diet on synthesis and utilisation of lipids for reproduction by the tsetse fly Glossina morsitans

Fatty acid composition of lipids from adult Glossina morsitans was unaffected by an in vitro fed diet of cow blood which induces production of under-sized offspring compared to that of flies fed on a superior diet of pig blood. The commonest fatty acids were C_16:0, C_16:1 and C_18:1 and only small differences in proportions were detected between virgin and pregnant females. Rate of lipid accumulation by males and females was the same and was unaffected by diet, but males achieved a maximum of 2.5 mg on day 9 while both virgin and fertilised females reached a maximum of 5,0 mg on day 14 of adult life. Lipid content of pregnant flies then fell to 3.0 mg on the day of larviposition and accumulation began again. A cow blood diet reduced the extent to which the lipids were utilised for larval growth and this was reflected in an altered secretory activity cycle in the female uterine gland. However, no effect on the growth of adult fat body was detectable in such flies. Mating and fertilisation, which influence reproductive events through activity of the endrocine system do not seem to affect the acquisition of lipid reserves by female Glossina. However, they clearly exert considerable influence over distribution of such reserves between fat body and uterine gland, which distribution is also affected by diet.     

Concordant Evolution of a Symbiont with Its Host Insect Species: Molecular Phylogeny of Genus Glossina and Its Bacteriome-Associated Endosymbiont, Wigglesworthia glossinidia

Many arthropods with restricted diets rely on symbiotic associations for full nutrition and fecundity. Tsetse flies (Diptera: Glossinidae) harbor three symbiotic organisms in addition to the parasitic African trypanosomes they transmit. Two of these microorganisms reside in different gut cells, while the third organism is harbored in reproductive tissues and belongs to the genus Wolbachia. The primary symbiont (genus Wigglesworthia glossinidia) lives in differentiated epithelial cells (bacteriocytes) which form an organ (bacteriome) in the anterior gut, while the secondary (S) symbionts are present in midgut cells. Here we have characterized the phylogeny of Wigglesworthia based on their 16S rDNA sequence analysis from eight species representing the three subgenera of Glossina: Austenina (=fusca group), Nemorhina (=palpalis group), and Glossina (=morsitans group). Independently, the ribosomal DNA internal transcribed spacer-2 (ITS-2) regions from these species were analyzed. The analysis of Wigglesworthia indicated that they form a distinct lineage in the γ subdivision of Proteobacteria and display concordance with their host insect species. The trees generated by parsimony confirmed the monophyletic taxonomic placement of Glossina, where fusca group species formed the deepest branch followed by morsitans and palpalis groups, respectively. The placement of the species Glossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on both the ITS-2 and the symbiont 16S rDNA sequence analysis, suggest that Glossina austeni should be placed into a separate fourth subgenus, Machadomyia, which forms a sister-group relationship with the morsitans group species. Received: 17 March 1998 / Accepted: 1 May 1998     

Physiological host range of two highly specialised mutualistic symbiotes: the fly Fergusonina turneri and its obligate nematode Fergusobia quinquenerviae,potential biocontrol agents of Melaleuca quinquenervia

In Australia, galls develop on Melaleuca quinquenervia (Cav.) S.T. Blake (Myrtaceae) as a result of the mutualistic association between the fly Fergusonina turneri Taylor (Diptera: Fergusoninidae) and its obligate nematode Fergusobia quinquenerviae Davies & Giblin-Davis (Tylenchida: Sphaerulariidae). The nematode induces gall formation, whereas the fly promotes gall maturation. Together they exploit M. quinquenervia buds and may inhibit stem elongation and flower formation. We delimited the physiological host range of this pair to determine their suitability as biological control agents of invasive M. quinquenervia populations in Florida, USA. Host use was assessed for eight species of Myrtaceae native to Florida, eight phylogenetically related ornamental species and oviposition alone on five non-myrtaceous species. Although oviposition was less specific, galls developed and matured only on M. quinquenervia. After establishment, galls are predicted to prevent flower and seed production, thereby reducing the regenerative potential of M. quinquenervia. This is the first example of an insect/nematode mutualism released as biological control agents of an invasive plant.     

Interspecific transfer of bacterial endosymbionts between tsetse fly species: infection establishment and effect on host fitness

Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.     

Sulphaquinoxaline in host diet as the cause of reproductive abnormalities in the tsetse fly (Glossina spp.)

Groups of females of Glossina austeni and G. morsitans morsitans fed on rabbits with 75 ppm sulphaquinoxaline and 7.5 ppm pyrimethamine in their diets had markedly lower fecundity than other groups fed on rabbits supplied with additive-free diets. There was little effect on the longevity of female flies. Females of G. austeni produced normal numbers of puparia for one reproductive cycle, large numbers of aborted larvae in succeeding reproductive cycles and many females eventually became sterile. The effect on G. m. morsitans was less marked but when the dose rate of the additives in the rabbit diet was quadrupled this species also showed greatly reduced fecundity, with no effect on longevity. In G. austeni, those puparia which were produced in later reproductive cycles were lighter and less viable than those produced in the first cycle.It is concluded that the toxicant in the rabbit diet was sulphaquinoxaline acting as a systemic insecticide but, at these low dose rates, at a sub-lethal level. The possible mode of action of sulphaquinoxaline and the significance of the results for the laboratory rearing of haematophagous arthropods are discussed.

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Résumé Des groupes de femelles de Glossina austeni et Gl. morsitans morsitans sont nourris sur des lapins, euxmêmes alimentés avec des aliments artificiels de type commercial, qui ont été ou non additionnés de composés chimiques coccidiostatiques, soit 75 ppm de sulfaquinoxaline et 7,5 ppm de pyrimethamine. Quand les glossines sont nourries sur des lapins ayant reçu l'aliment additionné des substances coccidiostatiques, la fécondité de G. austeni (en termes de production de pupes viables par femelle) est réduite de façon nette, bien que la longévité demeure inaffectée; G. m. morsitans se révèle moins sévèrement touchée.Dans les expériences conçues pour étudier l'effet de la sulfaquinoxaline et de la pyrimethamine, les lapins reçoivent un aliment en poudre auquel sont ajoutées des quantités appropriées de ces substances. Des groupes de femelles de G. austeni nourries sur des lapins ayant reçu un aliment contenant 75 ppm de sulfaquinoxaline et 7,5 ppm pyrimethamine se reproduisent normalement pendant un premier cycle reproducteur, après quoi elles engendrent un grand nombre de larves avortées et beaucoup de femelles deviennent éventuellement stériles. Les pupes produites par G. austeni au cours des cycles reproducteurs plus tardifs sont plus légères et moins viables que celles produites lors du ler cycle. Quand le taux des composés coccidiostatiques est quadruplé dans l'aliment des lapins, la fécondité de Gl. m. morsitans est aussi fortement réduite, sans effet sur la longévité. Quand Gl. morsitans est alimentée artificiellement sur du sang contenant 75 et 300 ppm. de sulfaquinoxaline, la fécondité des femelles est sérieusement réduite. Aucune différence ne se décèle quant aux effets relatifs de ces deux doses du composé coccidiostatique; il est possible que les mouches en absorbent une même petite quantité dans les deux cas, en raison de la faible solubilité du produit, qui n'est soluble que dans des solutions alcalines. La sulfaquinoxaline, aux faibles doses absorbées agit comme un insecticide systémique à des doses sub-létales. Ce coccidiostatique peut agir en interférant avec le métabolisme du folate au niveau des ovaires; les effets sur la fécondité deviennent progressivement plus marqués chez les femelles âgées et chez celles qui ont cessé de se reproduire on observe des ovaires atrophiés.L' incidence de ces faits sur les conditions d'élevage des Glossines, et d'autres arthropodes hématophages, est discuté.

     

Stability in an insect-pathogen model incorporating age-dependent immunity and seasonal host reproduction

A modified SIRS model is developed as a framework for the study of epizootiological dynamics in an insect-pathogen system. Linearized stability analysis reveals that the system with one immune and one susceptible host class can exhibit stable, periodic or unstable behavior depending on model parameters. In general, high pathogenicity, short pathogen propagule lifespan and high host reproductive rate are stabilizing influences. Pathogen transmissibility and propagule production/host do not influence local stability. The effect of seasonal host reproduction is studied because most insect hosts are seasonal in temperate climates. The basic stability dependence on model parameters holds except as modified by the length of the reproduction interval. The results of this study are compared with the recent work of Anderson and May. Scientific paper No. 82-7-179 of the Kentucky Agricultural Experiment Station, Lexington. This research has been financed in part with Federal funds from the USDA under grant number 82-CRSR-2-1000. The contents do not necessarily reflect the views and policies of the USDA.     

Effects of host age, host density and parent age on reproduction of the filth fly parasite Urolepis rufipes (Hymenoptera: Pteromalidae)

Urolepis rufipes Ashmead, a pteromalid wasp, was recently discovered parasitizing house fly and stable fly pupae in eastern Nebraska dairies. Studies have been conducted on the biology of this parasite to evaluate its potential as a biological control agent of stable flies (Stomoxys calcitrans (L.] and house flies (Musca domestica L.). House fly pupae were suitable as hosts for U.rufipes at all ages; however, significantly higher parasitism occurred on host pupae aged 96-120 h. Parasite-induced mortality (host mortality without progeny production) was higher than for other pteromalid parasites of filth flies under similar conditions. Parasitism increased with parasite--host ratio at 20 degrees C; however, the opposite was noted at 30 degrees C for parasite--host ratios ranging from 5:50 to 50:50. Fly eclosion decreased as parasite--host ratio increased at 20 degrees C, and no host eclosion occurred at the highest parasite--host ratios (20:50 and 50:50) at 30 degrees C. Females produced an average of 18.6 female and 7.6 male progeny. 88% of the progeny were produced during the first 6 days post parental eclosion. The short life span, low progeny emergence rate and high per cent host eclosion, in comparison with other parasite species, suggests that the Nebraska strain of U.rufipes may not an effective biological control agent of house flies.